Beta taipoxin as a cell growth factor and method

ABSTRACT

A peptide containing a sequence for the first fifteen amino acids from the N-terminal of Asn-Leu-Val-Glu-Phe-Gly-Lys-Met-Ile-Glu-Cys-Ala-Ile-Arg-Asn is used in a cell culture medium to promote cell growth in vitro and in the treatment of wounds. A method of preparation of the peptide is also claimed.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a division of application Ser. No.08/237,129, filed May 3, 1994, now the disclosure of which isincorporated by reference, and a division of application Ser. No.08/657,164, filed Jun. 3, 1996, now , the disclosure of which isincorporated by reference..

BACKGROUND OF THE INVENTION

[0002] The present invention relates to the discovery of non-toxic betataipoxin as a cell growth factor and a potent mitogen from poisonoussnake venom. Said composition consists of a polypeptide of beta taipoxinhaving molecular weight approximately 14,000 daltons and is free oftoxic effects.

[0003] Taipoxin as a whole intact molecule isolated from the venom ofthe Australian taipan Oxyuranus s. scutellatus is the most lethalneurotoxin. The whole molecule of taipoxin is a complex, composed ofthree subunits designated as alpha, beta and gamma, having molecularweight 45,6000 daltons.

[0004] Taipoxin can not be isolated by ion-xchange chromatography, sinceion exchangers tend to dissociate the active toxin complex. The majorlethality of taipoxin is due to the very basic alpha subunit. Themolecular weights of alpha, beta and gamma subunits of taipoxin are13,750, 13,473 and 18,354 daltons respectively.

[0005] To date numerous growth factors have been isolated from varioussources, and have been characterized To name a few, epidermal growthfactor (EGF), fibroblast growth factor (FGF), nerve growth factor OGF)and platelet derived growth factor (PDGF).

[0006] It is an object of this invention that the beta taipoxin as acell growth factor can be used to grow cells in serum free medium.Routinely, cells are grown in the presence of 5 to 20% fetal bovineserum (FBS) for research or production. The purification of productsderived from cells grown in serum containing medium is a cumbersome taskand furthermore, fetal bovine serum is the most expensive ingredient ofthe medium A mitogen like cell growth factor will provide serum freeenvironment with adequate cell proliferation.

[0007] A further object of this invention is to provide non-toxic betataipoxin having mitogenic activity as a composition for the promotion ofrapid burn and wound healing. In the case of cuts, surgical incisions,and abrasions to lessen the risk of infection and to shorten recoverytime.

SUMMARY OF THE INVENTION

[0008] Cell growth factor has been isolated as the beta subunit oftaipoxin by fractionating Oxyuranus s. scutellatus snake venom by highpressure liquid chromatography. After testing for mitogenic activity inall fractions, the mitogenic activity was revealed in one of thefractions. Cell growth factor is a beta taipoxin; which is a non-toxicfraction of snake venom and is a potent mitogen. Cell growth factor hasbeen isolated from the venom of the poisonous snake species Oxyuranus s.scutellatus.

[0009] The concentration of 0.1 μg/ml of the beta taipoxin non-toxicsubunit becomes a cell growth factor and in serum free medium it givesgrowth equivalent to medium containing 10% serum for a wide range ofcells. Cell growth factor is a beta taipoxin peptide having a molecularweight 14,000 daltons. In vivo experiments proved that beta taipoxin asa cell growth factor acts as a potent mitogen. The cut portion of mousehip skin healed much faster when cell growth factor was applied ascompared to the control counter part. Cell growth factor helped heal achronic foot wound of a friend. Cell growth factor is claimed as woundhealer and its use may be extended to treat burns.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 is a high pressure liquid chromatography profile ofOxyuranus s. scutellatus venom, resolved into 11 major fractions. Peaknumber 6 is representative of beta taipoxin.

[0011]FIG. 2 is a high pressure liquid chromatography profile ofconcentrated fraction number 6 containing beta taipoxin.

[0012]FIG. 3 is an electrophoresis profile of beta taipoxin on a 14%Novex gel, showing the molecular weight in the vicinity of 14,000daltons, when compared with Novex protein markers lysozyme, and trypsininhibitor, having molecular weights 14,400 and 21,500 daltonsrespectively on 14 % precast gel. Lanes 1 and 2 contain the cell growthfactor, lane 3 contains the markers.

DETAILED DESCRIPTION OF THE INVENTION

[0013] The cell growth factor consists essentially of a peptide of whichfirst 15 amino acid sequence is:Asp-Leu-Val-Glu-Phe-Gly-Lys-Met-Ile-Glu-Cys-Ala-Ile-Arg-Asn, which isexactly similar to beta taipoxin. For convenience this sequence isreferred herein as SEQ ID No: 1. It is believed that any peptide havingthe partial amino acid sequence SEQ ID No: 1 exhibits substantialutility as a cell growth promoter, a potent mitogen, regardless whetherit is synthesized or derived from natural sources. By the term, cellgrowth factor, we mean a substance whose presence produces a substantialnmitogenic effect on various types of cells, and that the mitogeniceffect is indicated, by an increase in cell growth or by a decreasedduplication time of various types of cells.

[0014] Preferably, the peptide cell growth factor contains the firstfifteen amino acids at N-terminal as given by SEQ ID No: 1, and has amolecular weight of about 14,000 daltons revealed by electrophoresiswhich is similar to beta taipoxin. In addition, cell growth factor iswater soluble and stable at 4 ° C. storage for its biological activity.Cell growth factor is stable at room temperature, 74 ° C. for severalweeks and its biological mitogenic activity is not altered by exposingit to ultra violet light overnight.

[0015] Cell growth factor, beta taipoxin, may be obtained essentially asa fraction of venom, from species of poisonous snake. Cell growth factoris preferably obtained from the venom of a species of Australian taipansnake, particularly the species Oxyuranus s. scutellatus.

[0016] The non-toxic beta taipoxin which is an active cell growth factoris obtained by separating the peptide fraction by, high pressure liquidchromatography, using ion exchange chromatography.

Fractionation of Venom

[0017] The active cell growth factor, a non-toxic beta taipoxin ispreferably separated from fresh frozen venom, although lyophilized wholevenom may also be used. The liquid venom is diluted 1:1 with 0,01 Mphosphate buffer saline (PBS) and preferably centrifuged to sedimentinsoluble debris, which can also be removed by filtration. Typically,the diluted and centrifuged 50 mg venom is loaded on high pressureliquid chromatography, from Toso Co. Japan and the ion exchange columnfrom Polymer Laboratories UK, maintained at 20 ° C. temperature. Aplurality of fractions according to the relative ionic charge are elutedpreferably, using gradientTnzma(2-amino-2-(hydroxymethyl)propane-1,3-diol)-HCl buffer pH 7.3. TheToso high pressure liquid chromatography automatically mixes 1.0 molarTriztna-HCL buffer with water to yield gradient Trizma-HCl buffer from0.01 molar to 1.0 molar. Any suitable gradient buffer may be used andTrizma-HCl buffer having pH 6.0 to 8.0 can be used.

[0018] The venom of Oxyuranus s. scutellatus resolved into 11 majorfractions by high pressure liquid chromatography (Drawing No. 1).Fraction 6 represents the active cell growth factor of beta taipoxin.The fraction containing the mitogenic active peptide may be used in thisform as a cell growth promoter, but may also be and preferably isfurther purified to obtain 100% purity to substantially remove mitogenicinactive substances as well. Preferably, the mitogenic active fraction 6is concentrated and dialyzed simultaneously using dialysis apparatusfrom Spectrum Co., to 1/20 volume and further purified by high pressureliquid chromatography as second run under identical conditions, such asgradient buffer temperature etc. The second time running of theconcentrate of the cell growth factor fraction 6 resolved into one peak(Drawing No. 2). This peak material is sequenced for its first fifteenamino acids from the N-terminal SEQ ID No:

[0019] 1 and was found to be exactly similar to beta taipoxin.

[0020] Initially each fraction was tested for the cell proliferativeactivity on rat adrenal pheochromocytoma, PC12, cells in concentrationsranging from 5 μg/ml to 0.1 μg/ml. PC12 cells were maintained inDulbecco Modified Eagle's Medium (DMEM) serum free medium and variousconcentrations of 11 fractions were added. The growth pattern of PC12cells in presence of each fraction was compared to the cells maintainedin DMEM containing 10% serum. All fractions showed toxic effect when theconcentration of the fraction was greater than 1 μg/ml. However,fraction 6 of Oxyuranus S. scutellatus venom showed proliferative growthof PC12 cells at 1, 0.5 and 0.1 μg/ml concentration.

[0021] The proliferative of cell growth factor was studied on wide rangeof cells of primary and secondary cultures. The results on primary cellcultures are shown in table 1. TABLE 1 Growth stimulating effect of cellgrowth factor on primary cultures of mouse kidney and spleen cellsversus conventional medium containing 10% FBS and serum free mediumMouse Kidney Mouse Spleen Medium 48 hours 96 hours 48 hours 96 hours 10%FBS ++ ++++ − ++ no FBS − + − − no FBS + 0.5 μg/ml +++ ++++ ++ ++ CGF noFBS + ++ ++++ ++ ++ 0.1 μg/ml CGF

[0022] TABLE 2 Growth stimulating effect of cell growth factor on PC12Cells* Incubation period Medium 48 hours 96 hours 10% FBS 2.3 × 10⁵ 4.3× 10⁵ Serum free 1.0 × 10⁵ 0.6 × 10⁵ serum free + toxic toxic 5 μg/mlCGF serum free + 1.9 × 10⁵ 2.1 × 10⁵ 1 μg/ml CGF serum free + 2.3 × 10⁵4.0 × 10⁵ 0.5 μg/ml CGF serum free + 2.4 × 10⁵ 4.3 × 10⁵ 0.1 μg/ml CGF

[0023] TABLE 3 Growth stimulating effect of cell growth factor onvarious established cell lines Serum free medium Cell Line none 0.1μg/ml 0.5 μg/ml 10% FBS Chang's liver retarded normal normal normal NIH3T3 retarded normal normal normal Skin no growth normal normal normalNS-20 retarded normal normal normal PC-12 retarded neurite neuritenormal SP/2 no growth no growth no growth normal

[0024] The results show that cell growth factor is nitogenic for most ofthe cells tested. Cell growth factor seems to be neurotrophic as PC12cells show neurite outgrowth in the presence of this growth factor.

[0025] Experimental cuts were made on both hips of test mnice. The rightcuts were treated with cell growth factor and the left with PBS. Thecuts treated with cell growth factor healed much faster.

[0026] A friend, experienced a deep wound on the top of his foot, whichshowed non closure after 3 months and was deemed to be chronic. Afterapplying cell growth factor, twice a day, his wound began to heal. Heexperienced the effect of cell growth factor within 24 hours.

[0027] Therefore, cell growth factor can be a wound healer.

[0028] Cell growth factor is stable at 4° C. for a long period of time.Cell growth factor is non-toxic, when 50 μg in 0.5 ml is injectedintraperitoneally in a 20 gram mouse. Being a cell growth promoterhaving a neurotrophic activity, this cell growth factor will be anexcellent wound and burn healer.

[0029] The cell growth factor is beta taipoxin of the present inventionis a particularly effective mitogenic agent. Since it is substantiallyfree of toxic activity, the cell growth factor can be used to grow widerange of cells in the serum free medium. Further, it may be used for arapid proliferation of cells either for research or for productionpurposes, also in the case of dermal monolayers to grow in vitro for thetreatment of bum victims and for wound healing.

[0030] It is further believed that cell growth factor, which is a betataipoxin, and its method of preparation of the present invention hasutility for treating bums and wounds including chronic wounds.Generally, the healing effects is accomplished by treating the wounds orcuts with cell growth factor having 100 μg/ml protein concentration inPBS in liquid form. It can be used after mixing with silvadene or anyother appropriate ointment. The application of cell growth factor onceor twice a day on the afflicted area, followed by dressing withsilvadeen, the healing effect starts within 24 hours. Moreover, afterthe wound is healed, no scar is produced because the hard dead tissue onthe wound sloughs off as a effect of cell growth factor.

Analysis of the Cell Growth Gactor Active Peptide

[0031] The concentrated fraction number 6 which was obtained by initialfractionating the crude venom, resolved into a single peak on secondtime fractionation on high pressure liquid chromatography underidentical conditions. The peak material was estimated to have amolecular weight in the vicinity of 14,000 daltons, when compared withNovex protein markers lysozyme, and trypsin inhibitor, having molecularweights 14,400 and 21,500 daltons respectively on 14% precast gel(Drawing No. 3).

[0032] The forgoing description of the invention is illustrative andexplanatory thereof, and many variations will occur to those in the art.It is intended that all such variations within the scope and spirit ofthe appended claims be embraced thereby.

1 1 1 15 PRT OXYURANUS SCUTELLATUS ISOLATE FROM VENOM 1 Asp Leu Val GluPhe Gly Lys Met Ile Glu Cys Ala Ile Arg Asn 5 10 15

What is claimed is:
 1. A culture medium characterized by the presence ofa peptide containing a sequence for the first fifteen amino acids fromthe N-terminal ofAsn-Leu-Val-Glu-Phe-Gly-Lys-Met-Ile-Glu-Cys-Ala-Ile-Arg-Asn identifiedin SEQ ID No:
 1. 2. A culture medium as in claim 1 further characterizedin that the peptide comprises beta taipoxin obtained from snake venom.3. A culture medium as in claim 2 further characterized in that the betataipoxin is obtained from the species Oxyuranus s. scutellatus, anAustralian taipan snake.
 4. A culture medium as in claim 3, furthercharacterized by the absence of serum.
 5. A culture medium as in claim 4further characterized in that the beta taipoxin has a molecular weightof about 14,000 daltons and is present at a concentration in the rangeof from about 0.1 μg/ml to about 1 μg/ml.
 6. A method for cultivatingvarious types of eukaryotic cells, said method comprising providing acell culture medium; placing cells in the cell culture medium; andcultivating said cells, characterized in that the cell culture mediumcontains a peptide containing a sequence for the first fifteen aminoacids from the N-terminal ofAsn-Leu-Val-Glu-Phe-Gly-Lys-Met-Ile-Glu-Cys-Ala-Ile-Arg-Asn identifiedin SEQ ID No:
 1. 7. A method as in claim 6 is further characterized inthat the cell culture medium is serum free and the peptide comprisesbeta taipoxin at a concentration in the range of 0.1 μg/ml to 1 μg/ml.